Genetic Analysis of Escherichia coli Biofilm Formation
Escheria coli was used in this model system to study the formation of biofilms. E. coli is able to form biofilms in several abiotic surfaces as long as nutrients are available. Furthermore, specific mutations were inserted into E. coli, which caused flawed biofilm formation. Half of these mutations affected normal flagellar function. It was found that the alleles fli, flh, mot and che dictate biofilm formation, and that it is dependent on motility not chemotaxis. Analysis of the mutants showed that motility is important for initial interaction with the abotic surface as well as for movement along the surface. Furthermore, type I pili is needed in order for E. coli to initially attach to any surface. Mannose can inhibit this attachment. What are biofilms? Biofilms are “matrix enclosed bacterial populations adherent to each other and/or to surfaces.” Cells stick to each other on an living and non-living surfaces, and they are embedded into a matric of extracellular polymeric substance (EPS). Biofilm EPS is also called slime, and it is a polymeric range made of extracellular DNA, proteins, and polysaccharides. Biofilms can form due to many reasons, such as cellular recognition of specific and non-specific attachment sites on certain surfaces and nutritional cues. Once a biofilm forms, the cell switches to a phenotypic shift in behavior. Many of the bacteria in the world live within biofilms, so it is important that we understand them and their development. They can be hundreds of microns in depth, which make them difficult to treat with antibiotics. Formation is also favored in nutrition sufficient environments, and the structures can be complex, including more than just the bacteria attached to the surface. Biofilms can also consist of one or more species. ''E. coli'' form biofilms in a nutrient-dependent fashion E. coli was used to study the initiation of biofilm formation. The microliter dishes that were used in the protocol were rinsed thoroughly to remove unattached cells and were stained with crystal violet. This showed that abiotic surfaces were not stained with CV, and motile laboratory strains of E. coli were able to adhere to different abiotic surfaces. In fact, E. coli formed biofilms on all the abiotic surfaces tested, which included PVC and polypropylene glass. Formation was influenced by the nutrition the environment provided. Formation initiated within two hours when E. coli was grown in LB. Additionally, biofilms were not able to form in minimal media. ''E. coli'' mutants defective in biofilm formation Mutants were screened to identify the genes required for biofilm formation in LB on PVC plastic. Mutants were selected on LB agar containing chloramphenicol. The chloramphenicol-resistant colonies were picked nad grown at room temperature in PVC dishes with glucose minimal medium. Candidate mutant strains were streaked for single colonies on LB agar and retested for their ability to form biofilms. All the insertion mutations showed defects and decreases in biofilm formation. Mutation Identification Mutant strains could affect biofilm formation for the following reasons: 1. Confers non-specific growth defect that indirectly affects biofilm development 2. Interferes in the formation without interfering with the growth rate Each of the mutants was analyzed for its ability to swarm on LB motility agar. About half of the mutants showed decreased ability. The rest were the same as the wild type. Most of the swarm mutants were severely defective in their ability to form biofilms. The rest of the mutants had less severe phenotypes. These mutants were analyzed via genetic linkage. A p1 lysate containing transposons was randomly inserted throughout the chromosome. This helped isolate a Tn10 that was linked to all the mutants. The precise locations of the mutations were identified by arbitrarily primed PCR and DNA sequence analysis. Eight locations were located in genes that encoded for the regulation of type I pili. Biofilm formation depends on motility, not chemotaxis. There are several reasons why flagella might be required. 1. Flagella could be directly involved in the attachment to abiotic surfaces and initiate biofilm formation 2. Motility could be needed to allow the bacterium to reach the surface 3. Motility might be required to help bacterial move along the surface and facilitate the spread and growth of the biofilm. Role of Type I pili Type I pili are critical for the initial attachment to abiotic surfaces. ''fim ''mutants are even more defective in initial attachment than cells that are paralyzed or do not have flagella. Most microscopic fields had no cells attached at all, which indicated that type I pili are required for initial interaction with abiotic surfaces. References http://onlinelibrary.wiley.com/doi/10.1046/j.1365-2958.1998.01061.x/full http://en.wikipedia.org/wiki/Biofilm